Upon insulin addition, GLUT4 transport vesicles accumulate and fuse at plasma membrane hot spots, creating clusters. To study the state of the GLUT4 molecules in these PM clusters in primary human adipose cells, we introduced a photo-switchable GLUT4 construct, HA-GLUT4-EOS, and applied a novel photo-activation localization microscopy technique, together with total-internal reflection fluorescence microscopy to track single GLUT4 molecules. We detected two distinct classes of GLUT4 molecule motions: unconstrained lateral diffusion and cluster-confined immobilization. We found that single GLUT4 molecules could get trapped in clusters, severely limiting their diffusion. Conversely, GLUT4 molecules were detected leaving their trapped state in these PM clusters (released) and resuming diffusion at 0.1 m2/s. Double-labeling of insulin-responsive vesicles with GLUT4-mCherry and IRAP-pHluorin was used to detect individual fusion events with PM. GLUT4 clusters were formed through fusion of GLUT4-containing vesicles with PM in an insulin-independent way. GLUT4 molecules were retained within the clusters by an unknown mechanism specific to GLUT4, but not to IRAP. Insulin, on the other hand, enhanced the rate of fusion events that released all GLUT4 into PM. These data provide the first evidence of a dynamic exchange of GLUT4 molecules between clusters and PM, and link insulin-dependent and insulin-independent GLUT4 recycling pathways. Moreover, these findings suggest that the amount of GLUT4 present in PM is not merely defined by the rates of exocytosis and endocytosis, but rather relies on GLUT4-specific molecular interactions at clusters that regulate its recycling. Thus we confirm a non-uniform GLUT4 distribution in the plasma membrane of primary human adipose cells and show the dynamic nature of GLUT4 organization into clusters. GLUT4 translocation to the plasma membrane (PM) is tightly controlled by insulin, and abnormalities in GLUT4 redistribution may contribute to the development of insulin resistance. Recently, GLUT4 clusters have been defined as PM domains that retain a fraction of GLUT4 on the cell surface. Our model for GLUT4 trafficking predicts that the fraction of clustered GLUT4 should increase with the total amount of GLUT4 in PM. In this study we investigate the significance of that link between the sequestration of GLUT4 into clusters and the insulin response of human adipose cells. We analyzed the insulin response in individual adipocytes isolated from fat biopsies obtained from a group of volunteers (7 women and 5 men, 27-67 yrs) that had a range of BMI of 27-49 kg/m2. The in vivo insulin sensitivity index (SI) was determined using the frequently sampled intravenous glucose tolerance test. In accordance with previous studies, subjects with a higher BMI (>35) showed a lower insulin sensitivity (SI <2), while the subgroup with BMI below 35 provided a wide range of SI from 0.15 to 7. Isolated adipose cells were transfected with HA-GLUT4-GFP and analyzed the next day. The amount of GLUT4 at the cell surface and the GLUT4 distribution into PM clusters were assessed by HA-antibody labeling and imaging of individual cells using confocal microscopy. We found for most patients that insulin stimulation increased total GLUT4 at the cell surface by up to four-fold. Both basal and insulin steady state levels of surface GLUT4 showed a positive correlation with SI. The density of clusters increased after insulin stimulation by 44.7 % (range of 17.0-98.3%), without a statistically significant correlation to SI. However, since the average normalized cluster intensity (molecules per cluster) increased with SI, clusters in general grew in size with increasing SI after insulin stimulation. Our data provide the first indication that both insulin sensitivity in individual subjects and GLUT4 trafficking in the adipose cells of those same subjects can be measured with success. The most insulin-resistant subjects have adipose cells whose GLUT4 increases minimally in the PM with insulin stimulation, but their GLUT4 clusters remain intact as quantitatively determined by intensity and density. As the SIs of subjects increase, the GLUT4 in their adipose cells increase in the PM, with a concomitant increase in clustered GLUT4 as predicted by our model, derived from the analysis of GLUT4 trafficking in rat adipose cells. Thus, the adipose cells of insulin-resistant subjects neither dismantle nor dramatically increase their fraction of clustered GLUT4.